Abstract Allogeneic Hematopoietic Cell Transplantation (allo-HCT) is an established therapy with curative intent for a variety of malignant and non-malignant disorders. Although overall survival rates are steadily improving (3), allo-HCT patients continue to incur significant morbidity and mortality from infections, GVHD and relapse. The overall objective of this P01 is to study both in mouse and man the mechanisms underlying these complications and develop novel therapeutic strategies to be tested in clinical trials in allo-HCT patients. The major themes of this P01 are: intestinal microbiota, antiviral resistance (especially to CMV), GVHD, antiviral NK and T responses, and adoptive T cell therapy. Our overall hypotheses are: 1)Specific members of the intestinal microbiota contribute to antiviral resistance, 2) The intestinal microbiota has a significant role in the development of GVHD, is a potent modulator of GVHD severity, and can be targeted to prevent or treat GVHD, 3) HLA-mediated efficiency of T-cell response shapes the NK repertoire and perturbations in anti-viral NK response will potentially affect the T-cell response, 4) Synthetic tumor-specific T cells can home to the tumor site and synthesize and release anti-neoplastic drugs locally with less toxicity and less tumor-resistance, 5) Third party EBV-specific cytotoxic T lymphocytes (EBV- CTL) can be used as ?off the shelf? adoptive cell therapy for EBV+ and ? malignancies in allo-HCT patients, and 6) Overall success of allo-HCT can be improved through clinical trials focused on TCRalpha/beta depletion of the allograft, third party EBV CD19 CAR T cells, and preservation of the intestinal microbiota through antibiotic stewardship and fecal microbiota transplantation. Our overarching Specific Aims are: 1) To correlate fecal microbiota composition with CMV reactivation or lower respiratory tract infection after allo-HCT and develop microbiota communities that can confer antiviral resistance in germ-free and antibiotic-treated mice, 2) To assess the role of the intestinal microbiota in the pathophysiology of acute and chronic GVHD and its use as a clinical biomarker, 3) To demonstrate that HLA genotype and activated NK cells can impact the T cell response to HCMV, as well as the T cell response to HCMV can shape the NK cell repertoire, 4) To generate and test Synthetic Enzyme Activating Killer (SEAKER) cells with prodrug/drug and killer switch systems and tumor-specificity through CAR and TCR mimic technology in preclinical and clinical settings, 5) To augment adoptive cell therapy with third party EBV-CTL for EBV+ and ? malignancies with epigenetic modifiers, CD19-CARs or checkpoint inhibitors, and 6) To initiate investigator-initiated clinical trials based on strategies developed in the individual Projects. This project is highly interactive with investigators at a single center, who have an extensive track record of collaborations and translation of their research into clinical trials in allo-HCT patients.